Abstract

Multiple Ca2+ channel β-subunit (Cavβ) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated Ca2+ channels, but the precise isoform expression pattern of Cavβ subunits in ventricular muscle has not been fully characterized. Using sequence data from the Human Genome Project to define the intron/exon structure of the four known Cavβ genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Cavβ isoforms. A total of 18 different Cavβ isoforms were detected in both canine and human ventricles including splice variants from all four Cavβ genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Cavβ subunit genes are expressed at the protein level, and the Cavβ subunits show differential subcellular localization with Cavβ1b, Cavβ2, and Cavβ3 predominantly localized to the T-tubule sarcolemma, whereas Cavβ1a and Cavβ4 are more prevalent in the surface sarcolemma. Coexpression of the novel Cavβ2c subunits (Cavβ2cN1, Cavβ2cN2, Cavβ2cN4) with the pore-forming α1C (Cav1.2) and Cavα2δ subunits in HEK 293 cells resulted in a marked increase in ionic current and Cavβ2c isoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Cavβ subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+ channels with distinct functional properties.

  • L-type calcium channel
  • splice variants

in the heart, L-type Ca2+ channels play an essential role in multiple cellular processes including cellular excitability and excitation-contraction coupling. Voltage-gated L-type Ca2+ channels are multimeric complexes consisting of a pore-forming α1-subunit and auxiliary subunits including β-, α2-δ-, and γ-subunits (9). Previous studies have demonstrated that the α1C-subunit (Cav1.2) is the major α1-subunit present in adult ventricular muscle (9), and multiple splice variants have been identified (38). Another, even greater source for diversity for L-type Ca2+ channels in the heart is the expression pattern of the auxiliary subunits which finely modulate the properties of the expressed channels.

The Cavβ subunit is a cytoplasmic protein that can be encoded by four different genes with multiple splice variants possible for each gene (6). The encoded proteins consist of five domains, and the two large central domains (D2 and D4) show high similarity between the gene products. However, the amino terminus (D1), small central linker (D3), and carboxy terminus (D5), exhibit much greater variability and are the sites for alternative splicing. The ultimate functional properties of the channel complex can be finely tuned depending on the Cavβ isoforms present. Cavβ subunit coexpression can shift the voltage dependence of channel activation and inactivation substantially (16, 55). Cavβ subunits also play an essential role in voltage-dependent facilitation of currents through L-type Ca2+ channels (10, 35). One of the most prominent and potentially important roles of Cavβ subunits is to act as a chaperone for trafficking Cavα subunits to the surface membrane, in part, by binding an endoplasmic reticulum retention signal (5, 12). In addition, there is emerging evidence that different β subunits may allow targeting to different subcellular domains (7, 13, 15, 40, 60). Therefore, the ultimate functional properties and subcellular localization of L-type Ca2+ channels are dependent on the particular auxiliary β-subunit isoforms present.

The Cavβ subunit isoform expression pattern in the heart has been evaluated in previous studies, but no clear consensus has emerged in the literature. The rat Cavβ2a isoform (GenBank accession no. M80545) was the first putative Ca2+ channel β-subunit identified in the heart (47), and it was generally believed that cardiac L-type calcium channels included primarily the Cavβ2a subunit (34, 47). However, the situation rapidly became more complex with the identification of multiple isoforms of the Cavβ1 gene in samples from human heart (14). More recently, the Cavβ3 gene has been found to be expressed in human heart (33). Differences between species may contribute to the confusion, but as additional studies have been performed even within a species consensus has not always emerged (63). Prior studies have often focused on defining the splice variants of a single Cavβ gene and have not evaluated for the full range of possible Cavβ isoforms. Although differential splicing of the four different Cavβ subunit genes has been observed in neuronal tissues (22, 41, 59), little work has focused on systematically identifying the expression profile for all Cavβ genes and their splice variants expressed in the human or canine heart.

The purpose of the present study was, first, to define the isoforms of the Cavβ subunits expressed in canine and human heart using an RT-PCR strategy made possible by sequence information from the Human Genome Project and published data. Secondly, we evaluated for differential subcellular localization of the different Cavβ isoforms by using isoform-specific antibodies. Finally, we determined the functional effects of previously uncharacterized Cavβ2c isoforms on heterologously expressed Cav1.2 channels. A preliminary report of these findings has been made (20).

MATERIALS AND METHODS

Genomic structure and similarity of Cavβ subunits.

Intron/exon structure of the four Cavβ subunit genes was identified by BLAST alignments of Cavβ cDNAs (GenBank) with the Human Genome Project draft sequence (Table 1). Similarity of individual exons from each Cavβ gene was determined by aligning the translations using PileUp [Genetics Computer Group (GCG), Accelrys, San Diego, CA], and the percent similarity was determined using the OldDistances program in GCG.

View this table:
Table 1.

Genomic structure of voltage-gated calcium channel Cavβ subunits

RNA preparation and cDNA synthesis.

Canine hearts were obtained using a protocol conforming to the Guide for the Care and Use of Laboratory Animals published by the National Research Council (1996). Cardiac myocytes were isolated enzymatically from adult canine left ventricle as previously described (28). mRNA was prepared from isolated canine cardiomyocytes using the FastTrack 2.0 system (Invitrogen, Carlsbad, CA). Human tissue was obtained from donor hearts rejected for transplant due to technical reasons, following a protocol approved by the University of Wisconsin Human Subjects Committee. Human total RNA was isolated from 1 g of left ventricular tissue using RNAzol B solution (Tel-Test, Friendswood, TX). Reverse transcription was performed on both canine mRNA and human total RNA using random hexamers and the SuperScript First-Strand Synthesis System (Life Technologies, Rockville, MD).

Polymerase chain reaction.

PCR primers were designed to identify all known splice variants of the Cavβ genes based on the published cDNAs and genomic structure from the Human Genome Project draft sequence. Oligonucleotide primers were synthesized by Life Technologies. Primer sequences are shown in Table 2, and primer pairs, amplicon sizes, and predicted protein lengths are shown in Table 3. All PCR experiments in canines were performed with cDNA synthesized from isolated left ventricular myocytes. Polymerase chain reactions contained 5.0 μl of cDNA from the reverse transcription reaction as template, 20 mM Tris·HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2.0 mM MgSO4, 0.1% Triton X-100, 0.1 mg/ml BSA; 2.0 μM each of dATP, dCTP, dGTP, and dTTP; 75 pmol of each primer, 5 U Taq Extender additive (Stratagene, La Jolla, CA), and 25 U Taq DNA polymerase (Fisher Scientific, Fair Lawn, NJ). PCR reactions were thermalcycled starting with an initial denaturation at 94°C for 3 min, cycled at 94°C for 45 s, 55°C for 45 s, and 72°C for 2 min for 45 cycles, and followed by 72°C for 7 min.

View this table:
Table 2.

RT-PCR primer sequences

View this table:
Table 3.

Isoform-specific RT-PCR primer pairs, amplicon sizes, amino acids amplified, and predicted protein length

Cloning and sequencing.

Amplified cDNA fragments were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining under UV light. Each fragment was then purified from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). cDNA fragments were cloned into pCR 2.1-TOPO using the TOPO TA Cloning method (Invitrogen). Cloned RT-PCR fragments were cycle sequenced using BigDye chemistry (Perkin-Elmer, Foster City, CA) and automated analysis (University of Wisconsin Biotechnology Center, Madison WI). Briefly, cycle sequencing was performed using 5.0 pmol T7 primer, 10 μl of cloned RT-PCR fragment in pCR 2.1-TOPO vector, 4.0 μl BigDye buffer, and 4.0 μl BigDye. Sequencing reactions were denatured at 95°C for 3 min and then cycled at 95°C for 20 s, 45°C for 30 s, and 60°C for 2 min for 35 cycles, and followed by 72°C for 7 min. DNA sequence identities were verified by aligning with previously cloned Cavβ subunits in GenBank and genomic sequences in the Human Genome Project using BLAST (1). Complete reading frames of Cavβ2cN1, Cavβ2cN2, Cavβ2cN4, and Cavβ2aN4 were amplified from human cDNA and cloned into pcDNA3.1HisTOPO (Invitrogen) using the following primers: fB2mik1v2 + rB2c and fB2c + rB2cterm.1 (Cavβ2cN1, GenBank accession no. AY393860), fB2N5v2 + rB2c and fB2c + rB2cterm.1 (Cavβ2cN2, AY393861), fB2UTR1.1 + rB2c and fB2c + rB2cterm.1 (Cavβ2cN4, AY393862), and fB2UTR1.1 + rB2aex7A and fB2aex7A + rB2cterm.1 (Cavβ2aN4, AY393859).

Cell culture and transfection of HEK 293 cells.

Low-passage HEK 293 cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM l-glutamine. HEK 293 cells were transfected with either Cav1.2 full-length rabbit cardiac subunit (43), except for alternative splicing in domain IV S3 (56) cloned into pGW1H (British Biotechnology, Oxford, UK), rabbit skeletal muscle Cavα2δ-1 (17) cloned into pGW1H, pSV40TAg to increase expression levels, and GFPpRK5 expressing the S65T bright green fluorescent protein mutant only or with Cavβ2cN1, Cavβ2cN2, Cavβ2cN4, or Cavβ2aN4 using the calcium phosphate transfection method (Invitrogen). Briefly, 10 μg of total cDNA were transfected into HEK 293 cells and incubated for 4 h. Cells were washed four times with PBS and incubated overnight in DMEM.

Electrophysiology.

Whole cell recordings were performed within 24 h after transfection. External solution consisted of (in mM) 10 BaCl2, 133 CsCl, and 10 HEPES (pH 7.4 with 1 N CsOH). Internal solution consisted of (in mM) 114 CsCl, 10 EGTA, 10 HEPES, and 10 Mg-ATP (pH 7.2 with 50 mM CsOH). Borosilicate glass pipettes were pulled to a resistance of 1–2.5 MΩ when filled with internal solution. Membrane capacitance and series resistance were compensated to at least 70%. Whole cell currents were recorded using an Axopatch 200B amplifier sampled every 40 ms and filtered through a low-pass filter at 5 kHz (Axon Instruments, Foster City, CA). Current-voltage (I-V) protocols consisted of a holding potential of −80 mV pulsing in steps of 10 mV to +70 mV for 50–400 ms and repolarizing to −80 mV. Leak and capacitive currents were subtracted using a P/4 protocol.

Whole cell conductance (G) was calculated from the peak inward IBa divided by the difference of the test potential and the estimated reversal potential of +60 mV. The G-V data were fit to a Boltzmann distribution according to the following equation Math where Gmax is the maximal whole cell conductance, V1/2 is the voltage midpoint for the distributions, and k is the slope factor. The data were fit using nonlinear least squares regression analysis with the Levenberg-Marquardt or Simplex methods available with Origin 6.0 (Microcal, Northampton, MA).

Fluorescence confocal microscopy.

Immunolabeling was performed on isolated canine left ventricular myocytes using the following primary antibodies: rabbit polyclonal antibodies to Cav1.2 (29), Cavβ1b antibody CW28 (58), Cavβ2 antibody CW48 (58), Cavβ3 (Alomone Labs, Jerusalem, Israel), and Cavβ4 antibody CW34 (58), and a guinea pig polyclonal antibody to Cavβ1a (61). Isolated myocytes were initially fixed with 2% buffered paraformaldehyde for 10 min. Fixed cells were permeabilized with Triton X-100 (0.1%) for 10 min and then quenched for aldehyde groups in 0.75% glycine buffer for 10 min. After washing with TBS (two 10-min washes), cells were incubated with 1 ml blocking solution (2% BSA and 2% goat serum, 0.05% NaN3 in TBS) for 2 h with gentle agitation at 4°C to block nonspecific binding. Subsequently, cells were incubated overnight with respective primary antibodies in blocking solution at 4°C. Antibody dilution of primary antibodies was 1:100 for the polyclonal anti-Cav1.2, anti-Cavβ1a, anti-Cavβ2, anti-Cavβ3, and anti-Cavβ4, and a 1:500 dilution of anti-Cavβ1b. Excess primary antibody was washed off with the use of blocking solution (three 1-h washes). The cells were then incubated overnight with Alexa-conjugated secondary antibodies (Molecular Probes, Eugene, OR; 2 mg/ml) diluted 1:200 in blocking solution. Highly cross-absorbed Alexa 568 goat anti-rabbit IgG (H+L) and Alexa 488 goat anti-guinea pig (H+L) were used. The cells were then washed with blocking solution (three 2-h washes), resuspended in blocking solution, and mounted on a coverslip. To determine nonspecific binding, control experiments with secondary antibody alone were also performed.

Imaging was performed with a Bio-Rad MRC 1024 laser-scanning confocal microscope equipped with a mixed gas (Ar/Kr) laser operated by 24-bit LaserSharp software (Bio-Rad, Hercules, CA). Image acquisition in the green channel utilized excitation at 488 nm with emission detected at 522 ± 17 nm. Acquisition in the red channel utilized excitation at 568 nm with emission detected at 605 ± 16 nm.

Membrane fractionation.

Sarcolemmal, T-tubular, and dyadic membrane fractions were prepared by the methods described previously (2). Briefly, portions of canine left ventricular tissue were homogenized and subjected to a series of differential centrifugations. Isolated canine left ventricular myocytes and human left ventricular tissue were also fractionated and analyzed on Western blots (data not shown). High-salt-washed membranes were layered on a discontinuous sucrose density gradient of 21, 31, 40, and 55% sucrose and centrifuged for 2 h at 141,000 gmax. Discontinuous density gradient centrifugation produced three distinct interfaces at 10/21%, fraction 1 (F1) enriched in surface sarcolemmal membrane; 21/31%, fraction 2 (F2) enriched in T-tubule membrane; and 31/40%, fraction 3 (F3) enriched in junctional complexes. Interfaces were pelleted at 141,000 gmax and suspended with the protease inhibitors 0.1 μg/ml leupeptin, 0.1 μg/ml pepstatin, and 1 μg/ml aprotinin and stored at −80°C. Protein concentrations were determined by the Lowry method.

SDS-PAGE and relative quantitative Western analysis.

Membrane proteins (60 μg) from each of the membrane fractions from both human and canine hearts were separated by SDS-PAGE using 7.5% bis-acrylamide gels as described by Laemmli (39). Membrane protein (20–60 μg of protein) was solubilized in sample buffer (62.5 mM Tris·HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.1% bromophenol blue) by warming to 60°C for 30 min prior to loading onto the gel. Following separation, proteins were transferred to nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) by blotting for 1 h at 105 V. Nonspecific binding sites were blocked by immersion of membranes overnight at 4°C in PBS detergent (0.1%, Tween-20) containing 5% (wt/vol) dried skim milk. Membranes were then probed with primary antibodies with the following dilutions: 1:500 for polyclonal anti-Cav1.2 and anti-Cavβ1b; 1:200 for anti-Cavβ2, anti-Cavβ3, and anti-Cavβ4; and 1:1,000 for anti-Cavβ1a. Donkey anti-rabbit immunoglobulin linked to horseradish peroxidase (1:50,000) detected bound antibody for Cav1.2, Cavβ1b, Cavβ2, Cavβ3, and Cavβ4. Goat anti-guinea pig immunoglobulin conjugated to peroxidase (Sigma, St. Louis, MO) was diluted 1:30,000 to detect Cavβ1a. Immunoreactivity was visualized using peroxidase-based chemiluminescent detection system, ECL (Amersham Life Sciences, Cleveland, OH). Relative quantitation of Western blots was accomplished using a Bio-Rad model GS-700 image densitometer. Conditions were optimized for each antibody by testing a range of antigen loading (20–120 μg of membrane protein) and determining the resulting densitometric signal. The relationship between antigen loaded and resulting densitometric signal was found to be linear over most of the tested concentration range. For all antibodies tested, at 60 μg of protein loaded, the signal was in the linear range. Multiple exposure times of the autoradiograms were also performed to optimize linearity and avoid signal saturation.

Statistics.

All values are presented as mean ± SE. Statistical significance was evaluated by the Student’s unpaired t-test. For multiple comparisons, analysis of variance (ANOVA) was performed. Differences with P < 0.05 were considered statistically significant.

RESULTS

Identification of Cavβ1 splice variants.

The results of the BLAST alignment of the Cavβ1 cDNAs to the human genomic DNA show that the Cavβ1 gene is composed of 15 exons of which only 14 are transcribed with exons 7A and 7B being alternatively spliced (see Table 1 and Figs. 1 and 4). This analysis is in agreement with the genomic structure of the Cavβ1 gene determined in a previous study using human genomic clones, with the exception of exons 13 and 14, which were described as alternative exons 13a and 13b previously by Hogan et al. (32). This minor discrepancy lies in the fact that our comparison of cDNA with the Human Genome Project sequence demonstrates that exons 13 and 14 are not alternative exons, and in the case of Cavβ1b, an alternative splice donor site in exon 13 is used to splice with exon 14.

Fig. 1.

RT-PCR of Cavβ1 splice variants in human and canine left ventricle. A: at top is a schematic of the 15 exons of the Cavβ1 gene, and below this are diagrams depicting the four splice variants amplified from canine and human left ventricle. The gray scale of each exon box denotes the similarity across the four Cavβ genes. Regions of greatest variability (6–34% similarity) are white, boxes shaded in light gray are exons of intermediate similarity (53–82%), and the boxes in dark gray mark exons with a high degree of similarity (71–100%). RT-PCR primers used to specifically amplify each splice variant are denoted with arrows. Alternatively spliced exons are denoted by peaked lines (“^”), and truncations are denoted by an asterisk (in Cavβ1d). Antibody epitopes are indicated by “Ab” above a bold line. B: RT-PCR products identifying each of the four splice variants with the actual size of amplified products of the Cavβ1 gene in human and canine left ventricle.

Initial screening for Cavβ1 transcripts was performed using primers flanking the alternatively spliced exon 7 using a forward primer in exon 3 (fB1) and a reverse primer in exon 8 (rB1) with mRNA prepared from isolated canine ventricular myocytes. Multiple fragments were amplified, isolated, and sequenced. These RT-PCR products were identified as Cavβ1a (exon 7A), Cavβ1b/Cavβ1c (exon 7B), and a novel splice variant that did not contain exon 7A or 7B, denoted Cavβ1d. The identification of Cavβ1d was difficult because the difference in the Cavβ1b/Cavβ1c and Cavβ1d products is only 20 bp, which is beyond the resolution of typical agarose gel electrophoresis, and thus the existence of Cavβ1d was only appreciated after screening and sequencing of multiple clones. Isoform-specific primers were then used to amplify Cavβ1a, Cavβ1b, and Cavβ1c, which were previously identified splice variants of the Cavβ1 gene (14, 48, 53), as well as the novel splice variant Cavβ1d (Fig. 1). The identity of all four Cavβ1 splice variant RT-PCR products was verified by sequencing. The Cavβ1a contains exon 7A, whereas Cavβ1b and Cavβ1c have exon 7B. In contrast, Cavβ1d skips exon 7A and 7B, which shifts the reading frame, resulting in a premature stop site in exon 8. As seen in Fig. 1A, both Cavβ1a and Cavβ1c have the same carboxy terminus ending after exon 13, but Cavβ1b uses an alternate splice site in exon 13 to which exon 14 is spliced to form the carboxy terminus. No splice variations were identified at the amino terminus of the Cavβ1 gene using specific NH2-terminal primers for RT-PCR. Additional experiments using total RNA isolated from human left ventricle also revealed the same four splice variants of the Cavβ1 gene with differential splicing restricted to exon 7 and the carboxy terminus at exons 13 and 14 (Fig. 1 and Table 1). Previous studies of human heart have also detected Cavβ1a, Cavβ1b, and Cavβ1c, but not Cavβ1d (14). Figure 4 shows the deduced amino acid sequences for the four identified Cavβ1 isoforms.

Identification of Cavβ2 splice variants.

The Cavβ2 gene has been suggested to encode the predominant Cavβ isoform(s) expressed in the heart; however, there is little information on the gene structure and extent of splice variants expressed. The genomic structure of the Cavβ2 gene was first determined by aligning the draft sequence from the Human Genome Project to the known Cavβ2 cDNAs in GenBank demonstrating 20 different exons (Table 1). The complex splicing of the Cavβ2 gene is evident by the five different amino terminal splice variants. We refer to these five different NH2 termini as N1 (exon 1A + exon 2A), N2 (exon 1B + exon 2A), N3 (exon 2B), N4 (exon 2C), and N5 (exon 2D). The middle of the gene is also spliced with four alternative splices with either exon 7A, exon 7B, a unique exon 7C, or no exon 7. In keeping with the current nomenclature of the β-subunits, these are termed as Cavβ2a (exon 7A), Cavβ2b (exon 7B), Cavβ2c (exon 7C), and Cavβ2d (no exon 7). Then to expand the nomenclature to include the NH2-terminal splice variants, we use the designations Cavβ2aN1, Cavβ2aN2, Cavβ2aN3,…etc. Thus the differential combination of the five possible NH2-terminal exons with the four different exon 7 splices yields at least 20 possible Cavβ2 splice variants.

Specific primers for RT-PCR were used to amplify splice variants of the Cavβ2 gene from canine and human heart as shown in Fig. 2. We have identified 9 of the possible 20 splice variants of the Cavβ2 gene present in both the human and canine heart: Cavβ2aN1, Cavβ2aN2, Cavβ2aN4, Cavβ2aN5, Cavβ2bN4, Cavβ2cN1, Cavβ2cN2, Cavβ2cN4, and Cavβ2dN4. Additionally, the Cavβ2bN4, Cavβ2cN1, Cavβ2cN2, Cavβ2cN4, and Cavβ2dN4 represent five novel isoforms not previously identified in any tissue. The Cavβ2aN4 has previously been identified in the rabbit and human heart (34, 57), Cavβ2aN2 in the rabbit heart (34), Cavβ2aN1 in the rat heart (63), and Cavβ2aN5 in mouse heart (42). The palmitoylated amino-terminal splice variants (Cavβ2N3) were not identified in either the human or canine heart. Also, no differential splicing was identified at the COOH terminus (from exons 8–14) of the Cavβ2 gene. Figure 5 shows the amino acid sequence alignments for the Cavβ2 splice variants identified in the human heart.

Fig. 2.

RT-PCR of Cavβ2 splice variants in human and canine left ventricle. A: at top is a schematic depiction of the 20 exons of the Cavβ2 gene, and below this the 9 splice variants detected in human and canine left ventricle are shown schematically with arrows underneath denoting primer pairs. Exons 10–12 are represented by “…”. The shadings of the exon boxes and other symbols are as described for Fig. 1. B: RT-PCR products amplified from the 9 splice variants with the actual size of amplified products transcribed from the Cavβ2 gene in human and canine left ventricle.

Fig. 3.

RT-PCR of Cavβ3 and Cavβ4 splice variants in human and canine left ventricle. A: schematic display of the 13 exons of the Cavβ3 gene with the detected single splice variant (arrows underneath denote primer pair), with Cavβ3b depicted along with the actual size of the RT-PCR product from canine and human left ventricle. B: comparison showing the 15 exons of the Cavβ4 gene, with the four lower diagrams corresponding to the four splice variants detected (arrows underneath denoting primer pairs). The shadings of the exon boxes and other symbols are as described for Fig. 1. The bottom of B shows actual sizes of the four splice variants amplified by RT-PCR.

Fig. 4.

Human Cavβ1 subunit amino acid sequence alignment (shaded regions identify regions of high similarity). Alignment show the Cavβ1 splice variants expressed in the heart including Cavβ1a (accession no. M92301), Cavβ1b (M92303), Cavβ1c (M92302), and Cavβ1d (AY393857).

Fig. 5.

Human Cavβ2 subunit amino acid sequence alignment (shaded regions identify regions of high similarity). Alignment shows Cavβ2 splice variants expressed in the heart with Cavβ2aN1 (AF423191), Cavβ2aN2 (AY393858), Cavβ2aN4 (AY393859), Cavβ2aN5 (AF423192), Cavβ2bN4, Cavβ2cN1 (AY393860), Cavβ2cN2 (AY393861), Cavβ2cN4 (AY393862), and Cavβ2dN4 (AY393863). *Cavβ2bN4 has been cloned only through exon 7B.

Identification of Cavβ3 and Cavβ4 splice variants.

We next determined whether isoforms of the Cavβ3 and Cavβ4 genes are expressed in the canine and human left ventricle. Previous studies have identified the intron/exon structure and the splice variants of both the human and murine Cavβ3 gene (44, 62). The results of the human genomic BLAST alignments to the human Cavβ3 cDNA show a similar intron/exon structure and with the same sized exons as previously shown in the human. Table 1 shows the 13 coding exons for the Cavβ3 gene from the Human Genome Project draft sequence (accession no. NT_029419.10). The numbering of the exons for the Cavβ3 gene is different from the other three Cavβ subunit genes because the Cavβ3 exon 2 is homologous with the exon 3 from Cavβ1, Cavβ2, and Cavβ4 genes. Thus the corresponding numbers for the homologous exons are shifted down so that exon 6 of the Cavβ3 gene is homologous to exon 7B in Cavβ1 and Cavβ2 and to exon 7 for Cavβ4 (Table 1). BLAST searches of this genomic clone do not identify homologous exons to either exon 7A in Cavβ1 and Cavβ2 or exon 7C in Cavβ2.

Utilizing isoform-specific primers, a single splice variant (Cavβ3b), containing all 13 exons of the Cavβ3 gene, was amplified from canine and human ventricle (Fig. 3A). This observation is consistent with other studies showing that the Cavβ3b isoform is expressed in human and rabbit heart and human brain (14, 33, 34, 44). Unlike the Cavβ1, Cavβ2, and Cavβ4 genes, the “d” variant, Cavβ3d, of the Cavβ3 gene was not detected in either canine or human heart. However, Cavβ3d has been previously identified in murine stem cells and more recently in the human heart (33, 44). RT-PCR screening of the amino and carboxy termini of the Cavβ3 gene did not detect additional splice variants.

The intron/exon structure of the human Cavβ4 gene has not previously been described. Table 1 shows the 15 coding exons for the Cavβ4 gene from the Human Genome Project draft sequence (accession no. NT_005403.13). BLAST searches of the Cavβ4 genomic clone did not identify homologous sequences to either exon 7A from Cavβ1 and Cavβ2 genes or exon 7C in Cavβ2. Isoform-specific primers were generated based on the human genomic alignments to the cloned Cavβ4 cDNAs. Initially, nonspecific Cavβ4 primers in exon 3 and specific primers to exon 7 and the junction of exons 6 and 8 were used to amplify Cavβ4b and Cavβ4d splice variants. Recent studies by Helton et al. (30) have shown that there are at least two NH2-terminal splices in the human brain including a novel short amino terminus (Cavβ4N2) that is homologous with the NH2 terminus of Cavβ3. Utilizing this information, we used specific primers to each Cavβ4 NH2 termini in conjunction with Cavβ4b- and Cavβ4d-specific primers to amplify four different isoforms of the Cavβ4 gene. Figure 3B shows the four splice variants of the Cavβ4 gene detected in canine and human ventricle: Cavβ4bN1 and Cavβ4bN2, which include exon 7; and Cavβ4dN1 and Cavβ4dN2, which are missing exon 7. Cavβ4bN1 was first cloned from the rat brain and subsequently in the human brain (GenBank accession no. U95020) (8). Subsequent studies have identified Cavβ4b in human cerebellum and temporal lobe using riboprobes and antibodies that are directed at the COOH terminus of Cavβ4b and thus do not differentiate between Cavβ4bN1 and Cavβ4bN2 (40, 59). However, no studies to date have identified Cavβ4bN1 in the human or canine heart. Studies by Hibino et al. (31) have identified by RT-PCR a truncated Cavβ4 splice variant in the chicken heart. Additionally, the Cavβ4 gene showed no differential splicing at the COOH terminus. Figure 6 shows the aligned amino acid sequences for the Cavβ3 and Cavβ4 isoforms.

Fig. 6.

Human Cavβ3 and Cavβ4 subunit amino acid sequence alignments (shaded regions identify regions of high similarity). Alignments show Cavβ3b (X76555) and Cavβ4bN1 (U95020), Cavβ4dN1, Cavβ4bN2 (AY054985), and Cavβ4dN2 splice variants expressed in the heart. *Human Cavβ4dN1 was cloned from NH2-terminal sequences specific for Cavβ4N1 starting in exon 2A through the termination codon.

Identification and subcellular localization of Cav1.2 and Cavβ proteins.

To verify that the Cavβ isoforms identified by RT-PCR were expressed as proteins, Western blot analysis and immunocytochemistry were performed with isoform-specific antibodies directed at Cav1.2, Cavβ1a, Cavβ1b, Cavβ2, Cavβ3, and Cavβ4. Furthermore, the subcellular distribution of these proteins was evaluated by semiquantitative Western blot analysis of different membrane fractions as well as by confocal microscopy of immunolabeled myocytes. Western blot analysis was performed on a membrane homogenate as well as three sucrose density gradient fractions enriched in surface sarcolemma (F1), T-tubular sarcolemma (F2), and junctional complexes (F3) (2). As shown on the Western blots in Fig. 7, the sucrose density gradients greatly enrich the membranes containing the Cav1.2 and Cavβ subunits relative to crude homogenate and provide more ready detection of these relatively low-abundance proteins.

Fig. 7.

Western blot and densitometric analysis of Cav1.2 and Cavβ subunits, displaying the results for a given subunit-specific antibody with a representative Western blot of discontinuous gradient fractions enriched in surface sarcolemma (F1), T-tubular sarcolemma (F2), and junctional complexes (F3), as well as membrane homogenate (H). The normalized, average data for densitometric scanning of each series of immunoblots are provided in the bar diagram (n = 3–7, mean ± SE; Hgt = homogenate). AF: antibodies directed at Cav1.2, Cavβ1a, Cavβ1b, Cavβ2, Cavβ3, and Cavβ4, respectively.

To determine the subcellular localization of the Cavα subunit in canine ventricle, immunoblots utilizing an antibody directed against Cav1.2 were performed. A doublet of 190 and 240 kDa was detected with the greatest abundance present in F2 (Fig. 7A), suggesting a strong T-tubular presence of this protein. This concurs with immunocytochemistry showing an ordered, punctate staining pattern typical of T-tubular staining (Fig. 8A). These results are also consistent with dihydropyridine binding (3H-PN200–110) of these fractions reported previously, with the greatest binding in the T-tubular fraction (2).

Fig. 8.

Subcellular localization of Cav1.2, Cavβ1a, Cavβ1b, Cavβ2, and Cavβ4 in canine left ventricular myocytes. Confocal images of isolated myocytes immunolabeled with primary antibodies directed against Cav1.2 (A), Cavβ1a (B), Cavβ1b (C), Cavβ2 (D), and Cavβ4 (E).

Western blot analysis and immunocytochemistry were then performed with Cavβ isoform-specific antibodies. First, we tested for the presence of Cavβ1a and Cavβ1b protein. Western blots probed with anti-Cavβ1a antibody showed staining of major bands at 58 and 64 kDa in all three enriched membrane fractions with the greatest abundance in F1 (Fig. 7B). This is similar to a 52/62-kDa doublet and a 71-kDa minor band described previously from crude homogenates of rabbit skeletal muscle where Cavβ1a is the predominant Cavβ subunit (61). However, the Cavβ1a was not detected at the protein level in rabbit heart using the same antibody (61). Immunocytochemistry with anti-Cavβ1a antibody revealed a distinct staining pattern compared with Cav1.2 staining, with predominantly surface sarcolemmal staining (Fig. 8B). In contrast, Western blots probed with anti-Cavβ1b antibody detected 66-kDa and 75-kDa proteins with the strongest signal in F2, the T-tubular enriched fraction (Fig. 7C). This pattern was quite similar to the proteins of 66 and 75 kDa specifically detected in HEK 293 cells transfected with Cavβ1b (GenBank accession no. M92303) cloned from the human brain (data not shown). This compares to an 82-kDa band detected in rat brain with this antibody (58) and a 75-kDa protein detected in canine brain (data not shown). The predicted size from the cDNA sequence is 66 kDa, suggesting possible differences in posttranslational processing between the tissues. The corresponding immunocytochemistry of Cavβ1b shows preferential staining of the T-tubules in canine cardiomyocytes (Fig. 8C).

Nine different isoforms of the Cavβ2 gene were identified using RT-PCR with expected protein molecular masses of 64–74 kDa. However, there are not specific antibodies available for each of these isoforms, so we utilized a Cavβ2 antibody that recognizes the common COOH terminus present on eight of the nine (Cavβ2dN4 is truncated and does not express the epitope). Anti-Cavβ2 antibody identified a predominant 75-kDa band on Western blots of all membrane fractions with the greatest signal from F2 (Fig. 7D). However, with longer exposure, Western blots revealed a range of sizes from 65–85 kDa. Previous studies have shown sizes of 62–100 kDa in canine, rabbit, porcine, and human heart and thus support the identification of multiple isoforms of the Cavβ2 gene (21, 25, 26, 51, 61). Longer exposures of blots also revealed a clear band in the crude homogenate lane not evident in the exposure on Fig. 7. Immunolabeled, isolated ventricular myocytes with anti-Cavβ2 showed a distinct T-tubule staining pattern as well as some staining of surface sarcolemma (Fig. 8D).

RT-PCR experiments have shown that a single isoform of the Cavβ3 gene was amplified in canine and human heart (Fig. 3A). The anti-Cavβ3 antibody identifies a 58-kDa protein in all three of the enriched membrane fractions. This is similar to other reports showing that a 58-kDa protein is expressed in rabbit heart and a 63-kDa protein in porcine heart (51, 61). Membranes from F2 showed the greatest Cavβ3 immunoreactivity, consistent with the highest abundance of the protein in T-tubule membranes (Fig. 7E). The anti-Cavβ3 antibody failed to give specific immunolabeling of isolated myocytes.

The results of the RT-PCR show the presence of four isoforms of the Cavβ4 gene, Cavβ4bN1, Cavβ4bN2, Cavβ4dN1, and Cavβ4dN2. The anti-Cavβ4 antibody used specifically recognizes an epitope on the COOH terminus of Cavβ4 and so would not be expected to detect Cavβ4dN1 or Cavβ4dN2. Western blots using anti-Cavβ4 antibody revealed a single 45-kDa band in canine membrane fractions. The predicted molecular mass based on the amino acid sequence for each Cavβ4 splice variant is 58 kDa (Cavβ4bN1), 22 kDa (Cavβ4dN1), 55 kDa (Cavβ4bN2), and 19 kDa (Cavβ4dN2). The Cavβ4 Westerns showed the greatest predominance of this protein by far in the surface sarcolemma-enriched F1 (Fig. 7F). With longer exposures of blots, bands were also seen in F3 and homogenates (data not shown). In agreement with the membrane fractionation studies, immunolabeling of the isolated myocytes with the anti-Cavβ4 antibody showed preferential staining of the surface sarcolemma (Fig. 8E). Previous studies have not detected Cavβ4 immunoreactivity in the adult ventricular muscle from rabbit (21, 61).

Experiments were also performed to verify the presence of these Cavβ proteins in human heart. Enriched membrane fractions were probed with the same panel of antibodies directed against Cavβ1a, Cavβ1b, Cavβ2, Cavβ3, and Cavβ4. Immunoreactivity was found specifically for each antibody with the identified proteins of comparable molecular weight to those found in canine heart (data not shown). The scarcity of human tissue made extensive membrane fractionation studies not feasible.

As a test to confirm that the immunoreactivity observed on Western blots was due to proteins present in cardiomyocytes, each antibody was tested on enriched membrane fractions made from enzymatically isolated canine ventricular myocytes. These preparations had no identifiable cells types except ventricular myocytes and should have minimal contamination. The panel of antibodies, likewise, recognized proteins of identical size to those detected in left ventricular tissue membrane preparations, confirming protein expression of all four Cavβ genes in ventricular myocytes (data not shown).

Electrophysiology of novel Cavβ2c isoforms.

The newly identified Cavβ2c isoforms are unique in containing the central exon 7C in contrast to exon 7A or 7B, and the functional properties of exon 7C containing Cavβ2 subunits have not previously been determined. Exon 7C is of particular interest, as homologous exons have not been found in any of the other Cavβ genes, unlike exons 7A and 7B, which share homology with the other Cavβ genes. Therefore, we isolated full-length human heart clones for Cavβ2cN1, Cavβ2cN2, and Cavβ2cN4 to compare with the well-characterized Cavβ2aN4 isoform. Heterologous expression experiments were performed in HEK 293 cells coexpressing Cav1.2, Cavα2δ, and Cavβ subunits using the whole cell patch-clamp technique. Representative raw current traces are shown for Cav1.2 + Cavα2δ and with each of the four cloned β-subunits in Fig. 9A. The peak current density was increased in the range of 6- to 10-fold by coexpression of Cavβ subunits with Cav1.2+Cavα2δ subunits as shown by the average I-V data in Fig. 9B. No significant differences between peak currents for the Cavβ isoforms were detected with ANOVA analysis of the group. The voltage dependence of current activation was shifted in the hyperpolarizing direction by coexpression of Cavβ subunits as shown by the negative shift of the peak of the I-V and more precisely shown by the activation curves calculated from the peak currents in Fig. 9C. Cavβ2aN4, Cavβ2cN2, and Cavβ2cN4 all resulted in a comparable hyperpolarizing shift of the V1/2 for the Boltzmann fit activation curves compared with Cav1.2+Cavα2δ only (−14.1 ± 0.9, −12.3 ± 1.1, or −13.1 ± 1.0 vs. −5.4 ± 0.5 mV, respectively, with P < 0.05 for each comparison). In distinction, coexpression of Cavβ2cN1 resulted in a significant shift in V1/2 (−8.3 ± 0.7 mV) compared with Cav1.2+Cavα2δ only, but this shift was significantly less than observed with the other Cavβ subunits studied. Thus alternative splicing limited to only the amino terminus of Cavβ2c can differentially impact voltage-dependent activation.

Fig. 9.

Functional effects of coexpressing Cavβ2c subunits with Cav1.2 and Cavα2δ. A: representative raw current traces at −30 mV, +10 mV, and +30 mV recorded from transiently transfected HEK 293 cells with Cav1.2 and Cavα2δ alone and with either Cavβ2cN1, Cavβ2cN2, Cavβ2cN4, or Cavβ2aN4. B: average current-voltage relationships, with inset showing subunit combinations and number of experiments. C: normalized whole cell conductance (G) vs. voltage (V) relationships with data fit by Boltzmann distributions as shown by the solid lines. D: average ratio of current decay determined at a test potential of +10 mV with the current measured at 50, 200, and 400 ms relative to the peak current for the different subunit combinations. *P < 0.05.

Cavβ subunits have also been previously demonstrated to have potent effects on channel inactivation. Therefore, we examined in detail current decay with and without Cavβ subunit coexpression. Inspection of the representative current traces (Fig. 9A) or the average data (Fig. 9D) revealed that, as has been the case for most Cavβ subunits, there is a significant acceleration of current inactivation. The ratio of the current remaining at 50, 200, and 400 ms relative to the peak current was used as a model independent measure of current decay. All four Cavβ subunits studied resulted in a significant acceleration of current decay relative to Cav1.2+Cavα2δ alone (P < 0.05 for each, Fig. 9D). There was no statistically significant difference among the Cavβ subunits themselves.

DISCUSSION

Using a systematic PCR screening strategy based on genomic sequence information made available by the Human Genome Project and published literature, we provide a detailed description of the rich diversity of Cavβ subunits expressed in human and canine ventricular muscle, with 18 distinct isoforms identified. Six of these isoforms have not previously been identified in the heart, and three full-length splice variants of the Cavβ2 gene (Cavβ2cN1, Cavβ2cN2, and Cavβ2cN4) were cloned and functionally characterized for the first time. We also detected expression of all four known Cavβ genes in the heart at the protein level using specific antibodies. Immunocytochemistry and membrane fractionation studies demonstrated distinctive patterns of subcellular distribution for different Cavβ isoforms with some more localized to the T-tubules, and others showing increased presence in surface sarcolemmal regions. Based on these results, we will discuss why this diversity is only now being fully appreciated and the implications for ventricular myocyte cell function.

Uncovering diversity of Cavβ isoforms.

The rich diversity of Cavβ isoforms in the heart may have been overlooked in prior studies for a number of reasons. First, early studies were limited by the information available on splice variants and known genes. Additionally, certain techniques, such as Northern blots, cannot easily distinguish between many of the splice variants of a single gene. By identifying the intron/exon structure using genomic and isoform alignments with GCG and the Human Genome Project BLAST, we were able to design PCR primers that would allow us to detect all known splice variants for the four Cavβ genes. At the protein level, the use of enriched membrane fractions improved the sensitivity of Western blots to detect relatively low-abundance Cavβ proteins. The present study focused on canine and human ventricle that have highly similar Cavβ isoform expression patterns, but other species, and particularly rodents such as mouse and rat, may have quite different Cavβ expression profiles. Because we identified all 18 splice variants in isolated canine ventricular myocytes by RT-PCR, we believe this indicates that these splice variants are expressed specifically in myocytes. However, it is impossible to rule out a very low level of contaminating cell types. Results using intact human and canine ventricular myocardium which contain endothelial cells, fibroblasts, smooth muscle and neurons interestingly revealed an identical pattern of Cavβ isoform expression suggesting either that no additional Cavβ splice variants are present in these cell types or significant contamination of the isolated myocyte preparations had occurred. Ultimately, the diversity of Cavβ subunits found in the heart may be even greater, as this study did not examine right ventricular or atrial tissue. Furthermore, distinct transmural patterns of Cavβ subunit distribution may also be present.

The presence of the unique palmitoylated Cavβ2aN3 in the heart has been controversial. It was first reported in the rat heart (47), but the probe used for Northern blot analysis was not specific for this Cavβ isoform. Several subsequent attempts have been unsuccessful in identifying this isoform in the heart of a variety of species included in the present study for human and canine heart (50, 63). In addition, a detailed cellular electrophysiology study comparing heterologously expressed β2a (Cavβ2aN3) and β2b (Cavβ2aN4) subunits with native calcium currents in rat ventricular myocytes argued that the palmitoylated Cavβ2aN3 was at least functionally absent in native ventricular myocytes, based on the kinetics of current decay (13). However, two recent studies have reported that the palmitoylated Cavβ2aN3 is expressed in the human heart (33, 64). Based on the majority of molecular and functional data, we conclude that Cavβ2aN3 is not expressed to a significant extent in human or canine ventricle.

Cavβ subunit nomenclature.

Finding and describing 18 isoforms of the Cavβ subunit required us to update the current β-subunit nomenclature (18). Unfortunately, the literature is complicated by a variety of naming schemes for different Cavβ subunit isoforms with many discrepancies. For example, the same group cloned “β2a” from the rabbit and also later identified “β2a” in the human heart, but these are different isoforms with distinct amino termini, Cavβ2aN4 and Cavβ2aN3, respectively (33, 34). As the list of splice variants has grown, particularly in the amino terminus of the Cavβ2 and Cavβ4, the existing nomenclature scheme is proving incomplete. We start by using the existing strategy of naming Cavβ subunit splice variants based on alternatively spliced exons 7A, 7B, and 7C in the central variable region (D3) of Cavβ2 protein by designating these isoforms by the gene number (2) followed by “a”, “b”, or “c”, referring to the alternative exon or “d” if no exon (i.e., Cavβ2a, Cavβ2b, Cavβ2c, Cavβ2d). We add to this a designation of the amino terminus structure using an intuitive description of the numerous splice variants based on the order that they occur within the intron/exon structure of the gene. Therefore, the inclusion of the first exon (designated “1A” in Fig. 2) in the transcribed message is denoted “N1” (i.e., Cavβ2aN1) and subsequent splicing patterns, as shown in Fig. 2, produce the other amino terminal variants (Cavβ2aN2, Cavβ2aN3, Cavβ2aN4, and Cavβ2aN5). This strategy has allowed us to uniquely identify each splice variant described in the present study and proves adequate to uniquely identify all of the currently known splice variants for Cavβ genes.

Subcellular localization of Cavβ subunits.

Cavβ subunits are known to play an important role in the membrane trafficking of Ca2+ channel complexes based largely on data from heterologous expression systems. For example, Cavβ subunits have been demonstrated to chaperone α1-subunits to the surface membrane when expressed in HEK 293 or Cos-7 cells (5, 12, 24, 36). There is also emerging evidence that different β-subunits may allow differential targeting to subcellular domains in certain cell types, such as when Cavβ subunits are heterologously expressed in a polarized epithelial tissue (7). In human hippocampus, a differential subcellular distribution of Cavβ isoform immunoreactivity has been detected with Cavβ1, Cavβ2, and Cavβ3 largely localized to neuronal cell bodies, whereas Cavβ4 showed a more dendritic localization (15, 40). Recently, studies have suggested differential localization of β-subunits in rat cardiomyocytes; however, these experiments detected exogenous expression of β-subunits by transduction of rat heart cells with adenoviral constructs with β1b, β2a (Cavβ2aN3), β3, and β4 fused with GFP (13, 60). The localization of exogenous β-subunits in the rat cardiomyocytes shows that the Cavβ1b-GFP is primarily present in the T-tubules, which is similar to what we have detected in canine cardiomyocytes. β2a-GFP (Cavβ2aN3) is localized to the surface sarcolemma, whereas in our study the Cavβ2 is primarily localized to the T-tubules with weak staining of the surface sarcolemma. This difference may reflect the use of the palmitoylated Cavβ2aN3 fused with GFP in the prior study with its unique membrane targeting properties (11, 50), compared with the present study detecting endogenous Cavβ2 isoforms which likely do not include Cavβ2aN3 as described above. Colecraft et al. (13) show that both Cavβ3-GFP and Cavβ4-GFP are intracellular with predominant fluorescence in the nucleus. In contrast, we detect native Cavβ3 and Cavβ4 in the sarcolemmal membranes in the canine cardiomyocytes. There are limitations in overexpressing exogenous proteins which can complicate the interpretation of such studies. For example, strong overexpression of the channel subunits may interfere with the normal protein trafficking of these isoforms, and competition with endogenous subunits may also complicate the results.

The present study for the first time provides evidence for differential subcellular distribution of endogenous Cavβ subunits in ventricular myocytes based on the combined results of membrane fractionation studies and immunocytochemistry. The Cavβ1b, Cavβ2, and Cavβ3 isoforms are preferentially localized to the transverse tubules with a weaker presence in the surface sarcolemma. Conversely, the Cavβ1a and Cavβ4 are preferentially localized to the surface sarcolemma and markedly less signal in the T-tubules. Regardless of the Cavβ subcellular targeting, we hypothesize that the majority of Cavβ isoforms colocalize with Cav1.2 subunits either at the surface membrane or in the T-tubules, but we did not do coimmunostaining experiments to rigorously verify this. Given the prominent role of L-type Ca2+ channels in the T-tubules in initiating excitation-contraction coupling, it is possible that Cavβ1b, Cavβ2, and Cavβ3 subunits importantly contribute to excitation-contraction coupling. Whether the Cavβ1a and Cavβ4 subunits contribute to alternative cell processes, such as cellular signaling, remains to be determined. Unfortunately, specific antibodies are only available for a minority of the Cavβ isoforms detected at the message level in this study, and so further refinement of the subcellular localization of many individual splice variants will require future study.

It is intriguing that two splice variants from the same Cavβ1 gene are localized differentially. Cavβ1a localizes primarily to the surface sarcolemma, whereas Cavβ1b preferentially targets to the transverse tubule sarcolemma. There are two main differences in these isoforms with differential splicing of the exons 7A (Cavβ1a) and 7B (Cavβ1b), as well as an additional exon 14 present only in Cavβ1b. The functional importance of alternatively spliced exons 7A and 7B may be connected to their close proximity to the “beta interaction domain” (BID) in the adjacent exon 8. Studies have shown that all known Cavβ subunits interact with Cav1 and Cav2 subunits through a high-affinity interaction site (BID) localized to a 30-amino acid region at the beginning of the D4 domain of the β-subunit (16). Thus alternative exons in this general region may alter the interactions with the α-subunits and potentially affect membrane trafficking. Alternatively, the distinct COOH termini of Cavβ1a and Cavβ1b may make for different subcellular targeting. Differential interactions of the Cavβ1 isoforms with cellular proteins other than the Cavα subunit likely play a major role in membrane trafficking and localization. The interaction with other proteins seems particularly possible for the Cavβ1 subunits given the proline-rich amino terminus homologous to a PDZ domain with an overall structure typical of the membrane-associated guanylate kinase (MAGUK) protein (27). The MAGUK protein family includes proteins such as PSD95 and can be important in targeting ion channels and membrane proteins (37). In addition, members of the RGK family of GTPases have recently been identified as interacting directly with Cavβ subunits, and these proteins have been shown to dramatically impact membrane trafficking of the channel complex (3, 19).

Functional impact of Cavβ structural diversity.

The remarkable diversity of the Cavβ subunits in the heart begs the question of the functional impact and cellular roles played by these distinct subunits. It has long been clear that β-subunit isoforms differ in their functional effects, as studied in heterologous expression systems and more recently in cardiomyocytes (13, 55). Many studies have described multiple functional effects of coexpression of Cavβ subunits with pore-forming Cavα subunits. These modulatory effects are limited to the Cav1 and Cav2 families of α1-subunits, not the low-voltage-activated Cav3 family. There are four general categories of effects: 1) changes in channel gating; 2) alterations in membrane trafficking and localization of channels; 3) regulation of channels by second messenger systems; and 4) alterations in drug block properties. For example, Cavβ subunit coexpression can shift the voltage dependence of channel activation and inactivation substantially, and these shifts vary in direction and magnitude depending on the Cavβ subunit studied. Another potential important difference between Cavβ subunits exists for the regulation of Cav1.2 channels by protein kinase A (PKA) that, in part, involves the specific phosphorylation of residues uniquely found in the carboxy terminus of the Cavβ2 subunit and not the other Cavβ subunits (23). Thus the different functional capabilities of each of the Cavβ subunit isoforms may allow for highly specific modulation of the L-type Ca2+ channel complex.

In the present study, we focused on exploring the functional properties of three novel splice variants Cavβ2cN1, Cavβ2cN2, and Cavβ2cN4, which we cloned from human heart and vary only in the amino terminus. The Cavβ2c subunits have previously been suggested, in part, at the message level by detection of alternative exon 7C (13, 52), but they have not previously been cloned in full-length or functionally characterized. When the Cavβ2c subunits were coexpressed with Cav1.2 and Cavα2δ in HEK 293 cells, we noted a large increase in expressed current compared with Cav1.2 + Cavα2δ channels that was likely due to both changes in channel gating and membrane trafficking of channel complexes as previously described for full-length Cavβ subunits (5, 12, 24, 46). Furthermore, differences in the precise modulation of gating by the Cavβ2c subunits were detected based on the smaller negative shift in the voltage dependence of activation (V1/2) when Cavβ2cN1 was coexpressed compared with Cavβ2cN2 and Cavβ2cN4. The Cavβ2cN1 isoform is unique among these isoforms in that it includes the proline-rich amino terminus encoded by exon 1A. Similarly, a recent study has demonstrated distinct effects on channel activation by Cavβ4bN1 and Cavβ4bN2 (same isoforms detected in human and canine heart here) which were attributed specifically to the proline-rich region in Cavβ4bN1 (30). In contrast, all three Cavβ2c isoforms accelerated channel inactivation similar to the Cavβ2aN4.

Takahashi et al. (57) recently studied all five NH2-terminal splice variants of Cavβ2 in the Cavβ2a backbone and likewise revealed NH2-terminal splice variant-specific effects on channel gating. The Cavβ2cN1, Cavβ2cN2, and Cavβ2cN4 splice variants that we evaluated all accelerated inactivation similarly to the matched subunits in the previous study: β2d (Cavβ2aN1), β2c (Cavβ2aN2), and β2b (Cavβ2aN4). In comparison, Takahashi et al. (57) found that β2a (Cavβ2aN3) and β2e (Cavβ2aN5) resulted in a relative slowing of inactivation, but we did not test these isoforms. The variations in voltage-dependent activation observed by Takahashi et al. (57) with the Cavβ2a amino terminal splice variants did not simply match with the shifts in voltage-dependent activation that we observed for the Cavβ2c isoforms, suggesting that the aggregate structure of both the amino terminus and the central variable region modulate activation. These recent results add to earlier work demonstrating the critical role of the amino terminus and central domain of the Cavβ subunit in finely regulating channel gating (45, 49).

Perhaps the most unique and functionally distinct family of Cavβ subunit isoforms will be the “d” isoforms, including the Cavβ1d, Cavβ2dN4, β4dN1, and β4dN2 isoforms identified in this report. As these isoforms skip exon 7, a frame shift results in a stop codon and the predicted protein is truncated, lacking the BID and the entire carboxy half of the protein present in other Cavβ isoforms. Recent studies have identified a β4c in the chick cochlea in agreement with our finding of a related isoform in human and canine heart. This protein was shown to be multifunctional in that it could not only modulate channel gating but also could target to the nucleus and regulate gene transcription (31). Another recent study has shown that β3trunc (Cavβ3d, not detected in the heart in our study) expression is increased in human ischemic cardiomyopathy, and expression with Cav1.2 and Cavα2δ in heterologous expression systems alters the open probability (Po) of the expressed L-type Ca2+ channel (33). Thus the “d” isoforms can still apparently interact with the channel but also may, in some cases, have important gene regulation functions.

Alternative splicing in the COOH terminus of Cavβ subunits may also have functional impact. Splicing of the COOH terminus is only well described for the Cavβ1 gene. In this case, the functional impact is most clearly seen in the essential role of the COOH terminus of Cavβ1a in voltage-dependent excitation-contraction coupling of skeletal muscle (4, 54). Whether the different COOH termini of the Cavβ1 subunits impact excitation-contraction coupling in cardiac muscle is unknown.

Overall, the remarkable diversity of Cavβ subunits expressed in the heart demonstrates the presence of multiple functionally distinct populations of L-type Ca2+ channels. The modular nature of the Cavβ subunit provides for alternative splicing that can precisely regulate channel gating, regulation, and localization. Thus subpopulations of Ca2+ channels may subserve distinct cellular functions. In addition, Cavβ subunits may participate in other cell processes such as gene regulation independent of the channel complex. We are only beginning to appreciate the roles of Cavβ subunits in cell biology.

GRANTS

This work was supported by National Institutes of Health Grants R01-HL-61537 and P01-HL-47053 (to T. J. Kamp), R01-HL-60723 (to C. T. January), and NRSA Postdoctoral Fellowship Award F32-HL-071476 (to B. P. Delisle).

Acknowledgments

The assistance of Thankful Sanftleben with manuscript preparation and of Jo Ellen Lomax with molecular biology is gratefully acknowledged. We also thank Kevin P. Campbell for providing the Cavβ1a antibody.

Footnotes

  • Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

    Address for reprint requests and other correspondence: T. J. Kamp, H6/343 Clinical Science Center, Box 3248, 600 Highland Ave., Madison, WI 53792 (E-mail: tjk{at}medicine.wisc.edu).

    10.1152/ physiolgenomics.00207.2003.

REFERENCES

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